Tissue-engineered prostheses such as replacement heart valves that utilize xenogeneic biomaterials as scaffold offer the possibility of a living implant that could resolve durability issues. In this application, xenogeneic biomaterials undergo "decellularization" to remove cell-associated antigens, thereby preventing tissue rejection. The assumptions of this approach are: 1) that important xenogeneic antigens are associated solely with the cellular component of the tissue, and 2) that disappearance of cells from histologic section equates to complete removal of antigens from the tissue. A likely and unfortunate consequence of these flawed assumptions was the recently reported catastrophic failure of Synergraft-decellularized non-fixed porcine heart valves in children. The application of xenogeneic biomaterials to fabrication of both glutaraldehyde-fixed and tissue-engineered bioprostheses could be enhanced by a better understanding of the immunogenicity of these materials. Critical to that approach is a better understanding of both the number and distribution (e.g. cell-membrane, extracellular matrix) of antigens within xenogeneic tissues. Thus, the specific aims of this proposal are: 1.To extract and separate by 2-D gel electrophoresis the cell membrane, cytoplasmic and extracellular matrix protein fractions of candidate xenogeneic biomaterials. 2. To determine antigenicity of xenogeneic biomaterials by exposing separated proteins to naturally-occurring and acquired antibodies generated against the candidate tissues. 3. To identify by MALDI-TOF-TOF mass spectrometry proteins that elicits an antigenic response. The proteomic approach described in this application offers a powerful and specific method for screening a large number of proteins for the critically important property of antigenicity across species. This information will provide important insight about the feasibility of xenogeneic tissue-engineered tissues and about xenotransplantation in general. Xenogeneic biomaterials are tissues from animals that could be used in the fabrication of replacement tissues for humans (i.e. tissue engineering). This application provides information about immune rejection of biomaterials that must be better understood before animal tissues can be used in tissue engineering. [unreadable] [unreadable] [unreadable]